Catalytic Binding Sites Research Articles

Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases.

The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake. The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane. The V-ATPases are composed of two functional domains. The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases. A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification. Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo. One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity. Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

Permethrin Carboxylesterase Functions as Nonspecific Sequestration Proteins in the Hemolymph of Colorado Potato Beetle

The inhibition profiles of the pI4.8 and 4.5 carboxylesterases from the permethrin-resistant strain of Colorado potato beetle were evaluated for a variety of inhibitors and insecticides. Both carboxylesterases were determined to be serine-hydroxyl-type esterases with different inhibition specificities toward phenylmethylsulfonyl fluoride, a serine-hydroxyl proteinase inhibitor, andpara-hydroxylmercuribenzoate, a cysteine-sulfhydryl group arylesterase inhibitor. Thepara-hydroxylmercuribenzoate-binding site appears separate from the catalytic binding site as determined byp-chloromercuribenzoate-agarose affinity chromatography of the purified pI4.5–4.8 carboxylesterases. The pI4.8 and 4.5 carboxylesterases, however, were not significantly different in their sensitivity to various insecticides. The levels of inhibition achieved by the insecticides and eserine showed a positive correlation with the hydrophobicity of the compounds, suggesting that the pI4.8 and 4.5 carboxylesterases can interact with a variety of hydrophobic compounds through nonspecific hydrophobic site(s). The kinetics of inhibition of the pI4.5–4.8 carboxylesterases elicited by permethrin and DDT are most similar to a mixed-noncompetitive type and a noncompetitive type of inhibition, respectively. Analysis of these kinetic differences indicates the presence of hydrophobic catalytic site(s) as well as hydrophobic noncatalytic site(s) that are available for the binding of esteratic (e.g., permethrin) and nonesteratic (e.g., DDT) hydrophobic insecticides, respectively. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI4.5–4.8 carboxylesterases suggests that resistance to permethrin is mainly conferred by sequestration. Sequestration of insecticides by the pI4.5–4.8 carboxylesterases appears to be nonspecific and associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides, such as other pyrethroids, DDT, and abamectin.

Investigation of a Catalytic Zinc Binding Site inEscherichia colil-Threonine Dehydrogenase by Site-Directed Mutagenesis of Cysteine-38

l-Threonine dehydrogenase catalyzes the NAD+-dependent oxidation of threonine forming 2-amino-3-ketobutyrate. Chemical modification of Cys-38 ofEscherichia colithreonine dehydrogenase, whose residue aligns with the catalytic zinc-binding residue, Cys-46, of related alcohol/polyol dehydrogenases, inactivates the enzyme [B. R. Epperly and E. E. Dekker (1991)J. Biol. Chem.266, 6086–6092; A. R. Johnson and E. E. Dekker (1996)Protein Sci., 382–390]. To probe its function, Cys-38 was changed to Ser, Asp, and Glu by site-directed mutagenesis. Mutants C38S and C38D were purified to homogeneity and found to be, like the wild-type enzyme, homotetrameric proteins containing one Zn2+atom per subunit. The circular dichroism spectra of these mutants were essentially identical to that of the wild-type enzyme. Mutant C38S was catalytically inactive but mutant C38D had a specific activity of 0.2 unit/mg, a level ∼1% that of the wild-type enzyme. After it was incubated with 1 mM Zn2+and then assayed in the presence of 15 mM Zn2+, mutant C38S showed only a trace of enzymatic activity (i.e., 0.013 unit/mg). Preincubation of mutant C38D with 5 mM Zn2+, Co2+, or Cd2+increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+halved and 0.5 mM Hg2+abolished activity. Zn2+-stimulated mutant C38D showed these properties: apparent substrate activation at low threonine concentrations, a maximum activity of 27 units/mg with 20 mM threonine, and inhibition by high levels of substrate; an activationKd= 3 mM Zn2+; and a pH optimum of 8.4 (in contrast to pH 10.3 for the wild-type enzyme). Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3-hydroxypentanoate, but Zn2+-activated mutant C38D is 10-fold more reactive with threonine than with 2-amino-3-hydroxypentanoate. In the absence of added metal ions, wild-type enzyme similarly uses substrates other than threonine and shows a dramatic increase in activity with only threonine when stimulated by either Cd2+or Mn2+; added Zn2+has no effect on activity with threonine. Cys-38 of threonine dehydrogenase, therefore, is located in an activating divalent metal ion-binding site. Having a negatively charged residue like Asp in this position allows the binding of a catalytic Zn2+ion which enhances activity with threonine and reduces activity with substrate analogs. Whether Cys-38 of wild-type threonine dehydrogenase binds a catalytic metal ion (possibly Zn2+)in vivoremains to be established.

Functional analysis of HIV-1 reverse transcriptase motif C: site-directed mutagenesis and metal cation interaction.

Motif C, present in all polymerases, has been proposed to be part of the catalytic and metal binding site of the enzyme, suggesting that polymerases have a common origin. Previously, we have shown that the metal ion manganese induces alterations in nucleotide substrate specificity in some polymerases. However, it is not known if the active site responsible for incorporation of nonspecific substrates is the same as that which incorporates specific ones. Here we show that manganese enables HIV-1 reverse transcriptase (RT) to incorporate rNTP's using RNA as a template, thus behaving as an RNA replicase. Also, we show that the mutation D186H in motif C strongly affects the natural DNA polymerase activity and that the RNA replicase activity becomes undetectable, suggesting that both activities depend on the same active site. This mutation changes the metal ion preference, with mutant RT presenting only 0.5% of the wild-type DNA polymerase activity in the presence of magnesium but 1.6% of the same activity in the presence of manganese. This variation in cation preference suggests that residue D186 is part of the metal binding site. Since residue D186 of motif C is essential for both activities and appears to be involved in the binding of an important cation needed for the specific activity, our results support the idea of a common origin for all polymerases, from an ancestral unspecified polymerase containing at least motif C.

Sequence and structure of the human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase heart isoform gene (PFKFB2).

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme that catalyzes the synthesis and degradation of Fru-2,6-P2, a key regulator of glycolysis. In mammals, several genes have been found to code for different PFK-2/FBPase-2 isoforms that differ in tissue distribution and enzymatic activities. In the present study, we report the characterization of the PFK-2/FBPase-2 heart isoform gene in humans (PFKFB2), including a full analysis of repetitive sequences and potential transcription binding sites. The genomic sequence of the PFKFB2 gene spans 22,485 bp and contains 15 exons. Heart cDNA analysis shows that PFKFB2 codes for a protein of 505 amino acids with a deduced molecular mass of 58,849 Da. Comparison of the human PFKFB2 gene to the homologous genes in rat and ox outlines a significant conservation of the intron-exon structure, sequence of 5' and 3' flanking regions, and simple sequence repetitive element positions. Most important, the human heart PFK-2/ FBPase-2 protein was found to retain all the important regulatory sites, as well as the catalytic and substrate binding sites identified in the rat and bovine heart isoforms, suggesting that the human enzyme is regulated in a manner similar to that observed in these organisms.

Mutagenesis of β-Glu-195 of the Rhodospirillum rubrum F1-ATPase and Its Role in Divalent Cation-dependent Catalysis

We introduced mutations at the fully conserved residue Glu-195 in subunit beta of Rhodospirillum rubrum F1-ATPase. The activities of the expressed wild type (WT) and mutant beta subunits were assayed by following their capacity to assemble into the earlier prepared beta-depleted, membrane-bound R. rubrum enzyme (Philosoph, S., Binder, A., and Gromet-Elhanan, Z. (1977) J. Biol. Chem. 252, 8742-8747) and to restore ATP synthesis and/or hydrolysis activity. All three mutations, beta-E195K, beta-E195Q, and beta-E195G, were found to bind as the WTbeta into the beta-depleted enzyme. They restored between 30 and 60% of the WT restored photophosphorylation activity and 16, 45, and 105%, respectively of the CaATPase activity. The mutants required, however, much higher concentrations of divalent cations and could not restore any significant MgATPase or MnATPase activities. Only beta-E195G could restore some of these activities when assayed in the presence of 100 mM sulfite and high MgCl2 or MnCl2 concentrations. These results suggest that the observed difference in restoration of ATP synthesis and CaATPase, as compared with MgATPase and MnATPase, can be due to the tight regulation of the last two activities, resulting in their inhibition at cation/ATP ratios above 0.5. The R. rubrum F1beta-E195 is equivalent to the mitochondrial F1beta-E199, which points into the tunnel leading to the F1 catalytic nucleotide binding sites (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). Our findings indicate that this residue, although not an integral part of the F1 catalytic sites, affects divalent cation binding and release of inhibitory MgADP, suggesting its participation in the interconversion of the F1 catalytic sites between different conformational states.

A photoaffinity probe covalently modifies the catalytic site of the cGMP-binding cGMP-specific phosphodiesterase (PDE-5).

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.

A novel dCMP methylase by engineering thymidylate synthase.

X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus caseiTS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 A further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10(11) change in specificity for methylation of dCMP versus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.

Organogenesis and angiogenin.

Our search for an angiogenesis-inducing factor in culture medium conditioned by human colon adenocarcinoma cells (HT-29) was inspired by the 'organizer' hypothesis originally postulated by Spemann. It led us to the isolation of angiogenin, a 14 kD protein homologous to pancreatic ribonuclease and one of the most potent stimulators of blood vessel formation known. This review summarizes the properties of angiogenin, its enzymatic and three-dimensional relationship to ribonuclease A (RNase A), those aspects of its structure that are critical for its biological function, and the therapeutic potential of angiogenin inhibition. Despite having the same arrangement of catalytic residues as RNase A, angiogenin has very low enzymatic activity. It lacks one of the four disulphide loops of RNase A; instead, the corresponding residues form part of a cell binding region. Both the catalytic activity and cell binding site are essential for angiogenesis. Angiogenin binds to cell-surface actin in confluent endothelial cells and to an as yet uncharacterized receptor on proliferating cells. Internalization and translocation to the nucleolus are also required for activity. Inhibitors of angiogenin can block angiogenesis in vitro and prevent tumour growth in vivo. Thus, a noncytotoxic neutralizing monoclonal antibody prevents the establishment of HT-29 human tumour xenografts in up to 65% of treated athymic mice. In those tumours that develop, the number of vascular elements is reduced. Actin also prevents the establishment of tumours while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse angiogenin. These antagonists also inhibit the appearance of tumours derived from two other human tumour cell lines. Inhibition of the action of angiogenin may prove to be an effective therapeutic approach for the treatment of malignant disease.

Effect of excess cadmium ion on the metal binding site of cabbage histidinol dehydrogenase studied by 113Cd-NMR spectroscopy

The enzymatic reaction of histidinol dehydrogenase (HDH) was stimulated by about maximally 75% on the addition of Cd 2+ ion to the reaction mixture. 113Cd-substituted HDH in the presence of excess Cd 2+ has been studied by 113Cd-NMR. 113Cd 2+ less than 1 equiv. per subunit preferentially binds to the catalytic metal binding site of the apoenzyme. Further addition of the metal ions causes the structural change of the enzyme including the catalytic metal binding site. HDH takes at least three discernible states, which may correspond to the more or less active forms of the enzyme induced by metal ions.

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E.

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 A resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.

Site-directed Mutagenesis of the Yeast V-ATPase A Subunit

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.

Asymmetry of catalytic but not of noncatalytic sites on Escherichia coli F1-ATPase in solution as observed using electron spin resonance spectroscopy.

We have employed electron spin resonance (ESR) spectroscopy using different spin-labeled nucleotides to probe the environment of nucleotides bound at catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1-ATPase. We found that nucleotides bound in the noncatalytic binding sites were strongly immobilized and resulted in ESR spectra with one single corresponding spectral component. Nucleotide bound at the catalytic binding sites gave rise to two different signals in the ESR spectra indicative of two distinct conformations of the catalytic sites of the protein. One conformation of the catalytic sites is very tight, resulting in signals identical to those of the noncatalytic sites, while the second type of catalytic sites permitted an unusually high mobility of the bound spin-labeled nucleotide. The findings are compared to the requirements of the binding change mechanism and to the features of the nucleotide binding sites as elucidated from the X-ray structural model of the beef heart mitochondrial enzyme.

Aluminum interaction with plasma membrane lipids and enzyme metal binding sites and its potential role in Al cytotoxicity

The trivalent cation aluminum can cause chronic cytotoxicity in plants, animals and microorganisms. It has been suggested that Al interaction with cell membranes and enzyme metal binding sites may be involved in Al cytotoxicity. In this study, the binding of Al to microsomes and liposomes was found to be lipid dependent with the signal transduction element phosphatidylinositol-4,5-bisphosphate having the highest affinity for Al with an Al:lipid stoichiometry of 1:1. Al binding was only reduced in the presence of high concentrations of Ca 2+ (>1 mM). Both citrate and, to a lesser extent, malate were capable of preventing Al lipid binding, which is consistent with the involvement of these organic acids in a recently described Al detoxification mechanism in plants. The effects of AlCl 3, Al-citrate and ZnSO 4 on metal-dependent enzyme activities (enolase, pyruvate kinase, H +-ATPase, myosin, Calpain, proteinase K, phospholipase A 2 and arginase) was assayed in vitro. While Zn 2+ was capable of inhibiting all the enzymes except the H +-ATPase, AlCl 3 and Al-citrate had minimal effects except for with phospholipase A 2 where an interaction with AlCl 3 occurred. However, this could be negated by the addition of citrate. The results indicate that, contrary to current hypotheses, the toxic mode of Al is not through an interaction with enzymatic catalytic metal binding sites but may be through the interaction with specific membrane lipids.

Structures of catalytic and anti-peptide antibody binding sites : Ian A. Wilson, Robyn L. Stanfield, Matthew R. Haynes, Jayant B. Ghiara, Hongmei Li, Andreas Heine, Enrico A. Stura, The Scripps Research Institute, Department of Molecular Biology, 10666 North Torrey Pines Road, La Jolla, California 92037, USA

Structures of catalytic and anti-peptide antibody binding sites : Ian A. Wilson, Robyn L. Stanfield, Matthew R. Haynes, Jayant B. Ghiara, Hongmei Li, Andreas Heine, Enrico A. Stura, The Scripps Research Institute, Department of Molecular Biology, 10666 North Torrey Pines Road, La Jolla, California 92037, USA

Site-directed mutagenesis of putative catalytic and nucleotide binding sites in N10-formyltetrahydrofolate synthetase

To determine the importance of specific amino-acid residues in catalysis and substrate binding by N 10-formylH 4folate synthetase, one lysine and three histidine residues in the enzyme from Clostridium cylindrosporum were mutated to glutamine and serine residues, respectively. These residues, Lys-71, His-125, His-131, and His-268, are conserved in four bacterial and five eukaryotic proteins for which the amino-acid sequences are known. Previous evidence indicated that a histidine residue may play a role in catalysis and it has been proposed that Lys-71 could be a member of a putative nucleotide binding consenus sequence. The histidine mutations, H125S, H131S, and H268S, produced proteins that were unstable and were proteolytically degraded to different extents. No activity of purified H268S could be detected and the 240 kDa native tetramer was also absent. Activities of the H125S and H131S mutants could be measured and the K m values of the substrates were similar to those for the wild-type enzyme. It is concluded that the mutations resulted in monomers that do not fold properly and/or do not associate to the active tetramer and, as a consequence, are susceptible to intracellular proteolytic digestion. On the other hand, the K71Q mutation did not produce proteolyzed material. The resulting protein had a k cat value which was reduced by a factor of 3.3 × 10 −4. K m values of the substrates were not affected, nor were the affinty constants for MgATP and H 4PteG 3. CD and fluorescence spectra demonstrated that little change in the tertiary structure of the protein had occurred as a result of the mutation. The monomer form of K71Q was less stable than the monomer of the wild-type enzyme and reassociated less efficiently than the wild-type. From these results it is suggested that Lys-71 plays a critical role in catalysis by N 10-formylH 4folate synthetase and that this residue may reside at an intersubunit interface.

F 1F 0-ATP synthase: development of direct optical probes of the catalytic mechanism

Using strategically-placed tryptophan (Trp) residues as optical probes to monitor nucleotide binding and hydrolysis, we demonstrate that all three catalytic nucleotide binding sites in F 1-ATPase must be filled to obtain physiological ( V max) MgATP hydrolysis rates. At V max hydrolysis rates, the predominant enzyme species has one of the three catalytic sites filled with unhydrolyzed substrate MgATP, the other two sites are filled with product MgADP. A specifically-inserted Trp probe was also developed to characterize nucleotide binding to the noncatalytic sites, and a model to explain the specificity of these sites is shown. These sites appear to play no role in ATP hydrolysis.

An Attempt to Convert Noncatalytic Nucleotide Binding Site of F1-ATPase to the Catalytic Site: Hydrolysis of Tethered ATP by Mutated α Subunits in the Enzyme

The α and β subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the β subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the α subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the α subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the β subunit with Gln. The resultant mutant α3β3γ complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant α3β3γ complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding site was hydrolyzed, indicating that a primitive catalytic ability was generated at the α subunit by the introduced Glu.

Patterns of Cross‐Tolerance to Herbicides Inhibiting Acetohydroxyacid Synthase in Commercial Corn Hybrids Designed for Tolerance to Imidazolinones

Two commercial corn (Zea mays L.) hybrids, ICI 8532 IT and Pioneer 3180 IR, designed for tolerance to imidazolinones, were evaluated for tolerance to various acetohydroxyacid synthase‐inhibiting herbicides at the whole plant and enzyme levels. The purpose was to establish and compare the underlying enzymatic basis for tolerance in these plants, which were produced by contrasting methods and contain different genetic complements of the altered target enzyme. ICI IT plants exhibited significant tolerance (40‐fold higher rates for 50% inhibition of growth than the control hybrid) to imazethapyr, and somewhat less to imazaquin and pyrimidyloxybenzoate. ICI IT plants were no more tolerant to chlorsulfuron or flumetsulam than the control hybrid. Pioneer IR plants were highly tolerant to all of the AHAS inhibitors tested, requiring rates 200‐ to 2000‐fold higher than those of the control hybrid for 50% inhibition of growth. The basis for tolerance was evaluated on acetohydroxyacid synthase (AHAS, EC 4.1.3.18) in vitro. Relative to unmodified AHAS isolated from the control hybrid, AHAS isolated from ICI IT corn was seven‐fold less sensitive to imazethapyr, five‐fold less sensitive to pyrimidyloxybenzoate and two‐fold less sensitive to imazaquin, but was as sensitive to chlorsulfuron and flumetsulam. AHAS from Pioneer IR corn was highly insensitive to all of the AHAS inhibitors tested. Sensitivity to the feedback inhibitor leucine was not detectably altered in the modified enzymes. Substrate saturation kinetics of AHAS isolated from ICI IT corn were identical with those of unmodified AHAS, while AHAS from Pioneer IR required a two‐fold higher concentration of pyruvate for half‐maximal saturation. The results support the theory that because the catalytic and herbicide binding sites of AHAS are distinct from each other, crops can be designed for tolerance to AHAS‐inhibiting herbicides with little effect on performance.